Effects of Roxadustat on Erythropoietin Production in the Rat Body

Anemia is a major complication of chronic renal failure. To treat this anemia, prolylhydroxylase domain enzyme (PHD) inhibitors as well as erythropoiesis-stimulating agents (ESAs) have been used. Although PHD inhibitors rapidly stimulate erythropoietin (Epo) production, the precise sites of Epo production following the administration of these drugs have not been identified. We developed a novel method for the detection of the Epo protein that employs deglycosylation-coupled Western blotting. With protein deglycosylation, tissue Epo contents can be quantified over an extremely wide range. Using this method, we examined the effects of the PHD inhibitor, Roxadustat (ROX), and severe hypoxia on Epo production in various tissues in rats. We observed that ROX increased Epo mRNA expression in both the kidneys and liver. However, Epo protein was detected in the kidneys but not in the liver. Epo protein was also detected in the salivary glands, spleen, epididymis and ovaries. However, both PHD inhibitors (ROX) and severe hypoxia increased the Epo protein abundance only in the kidneys. These data show that, while Epo is produced in many tissues, PHD inhibitors as well as severe hypoxia regulate Epo production only in the kidneys.

We therefore hypothesized that, if these drugs increase Epo by targeting the prolylhydroxylase domain enzyme outside the kidneys, then this extrarenal increase in HIF1α/2α expression might induce unwanted side effects. Thus, a long-term increase in HIF1α/2α expression outside the kidneys may invoke other diseases. As such, the purpose of this study was to determine the tissue(s) in which Epo production increases with prolylhydroxylase domain enzyme inhibitor (ROX) administration. To accomplish this goal, we developed a novel assay that employs immunoblots of deglycosylated tissue samples, and this enables Epo protein abundance to be quantified over a wide range.
The hematopoietic effects of PHD inhibitors on renal anemia in chronic kidney disease (CKD) patents have been established [9,26,27]. Whether PHD inhibitors stimulate Epo production in the liver or not is the major question. Severe hypoxia and PHD inhibition are known to stimulate Epo mRNA expression not only in the kidneys but also in the liver [28][29][30]. Kato et al. reported that TP0463518, a PHD inhibitor, stimulated Epo mRNA expression in the liver but not in the kidneys in 5/6 nephrectomized rats [29], suggesting that TP0463518 increases Epo production in the liver.
Moderate hepatic impairment did not affect the hematopoietic effects of Roxadustat (ROX), a PHD inhibitor, suggesting a small role of the liver for Epo production by ROX [31]. A major problem in these studies was the lack of Epo protein determination. They examined the Epo mRNA and plasma Epo concentrations to evaluate the Epo protein production. The liver produces Epo in utero but then stops at birth through Epo gene methylation [32][33][34]. These data suggest that, within the liver, Epo transcription occurs but does not result in protein translation.
To test this hypothesis, a better approach to quantifying Epo abundance in tissue and in plasma is required. To accomplish this goal, we developed a method for quantifying Epo tissue that employs Western blot analysis and does not require pre-purification of the samples [35,36]. We observed that Epo deglycosylation with PNGase increases Epo assay sensitivity~10 fold. Using this Epo assay, we investigated which tissues increase Epo production in response to the PHD inhibitor, ROX or in response to severe hypoxia.

Comparison of Glycosylated and Deglycosylated Epo Protein by Western Blotting
We compared the glycosylated and deglycosylated Epo protein abundance using Western blotting. ROX-treated kidney lysates were deglycosylated with or without PNGase and were examined by two-fold serial dilution. As shown, the threshold of detection was lower for the deglycosylated compared with for the glycosylated protein ( Figure 1).

Western Blot Analysis of ROX-Induced Epo Production in the Body
ROX-induced Epo production in the body was examined using deglycosylated Epo. Epo production in ROX-treated male rats was observed most abundantly in the kidneys (Figure 2a). Low levels of Epo protein abundance were seen in the salivary glands, pancreas and epididymis. Therefore, we tested whether ROX stimulates Epo production in these tissues by comparing the Epo abundance by immunoblot of lysates from control and ROX-treated rats.
We observed, however, that Epo protein abundance in the salivary glands, pancreas, and epididymis were unchanged with ROX treatment (Figure 2b,c). Epo protein expression was not detected in the liver from either control or ROX-treated rats (Figure 2a). ROX treatment increased the Epo protein abundance only in the kidneys (Figure 2b). The effect of ROX on Epo protein expression was also examined in female rats. Small expressions were observed in the salivary glands, thymus, spleen and ovaries. ROX did not increase the expression in these organs (Figure 2d,e). The increase of Epo protein expression was observed only in the kidneys, which was the same for males. Our findings are summarized in Figure 2f.

Western Blot Analysis of ROX-Induced Epo Production in the Body
ROX-induced Epo production in the body was examined using deglycosylated Epo. Epo production in ROX-treated male rats was observed most abundantly in the kidneys (Figure 2a). Low levels of Epo protein abundance were seen in the salivary glands, pancreas and epididymis. Therefore, we tested whether ROX stimulates Epo production in these tissues by comparing the Epo abundance by immunoblot of lysates from control and ROX-treated rats.
We observed, however, that Epo protein abundance in the salivary glands, pancreas, and epididymis were unchanged with ROX treatment (Figure 2b,c). Epo protein expression was not detected in the liver from either control or ROX-treated rats (Figure 2a). ROX treatment increased the Epo protein abundance only in the kidneys (Figure 2b). The effect of ROX on Epo protein expression was also examined in female rats. Small expressions were observed in the salivary glands, thymus, spleen and ovaries. ROX did not increase the expression in these organs (Figure 2d,e). The increase of Epo protein expression was observed only in the kidneys, which was the same for males. Our findings are summarized in Figure 2f.

Epo Production during Severe Hypoxia (In Vivo)
The next series of experiments examined the effect of severe hypoxia on Epo production in various tissues taken from male and female rats. In response to hypoxia, the Epo protein abundance increased dramatically in the kidneys from both male and female rats (Figure 3b,c,e). A low level of Epo protein was detected in the salivary glands, pancreas, spleen and epididymis or ovaries in males and females, although the Epo protein abundance was unchanged in these tissues in response to severe hypoxia (Figure 2a-e). Our findings are summarized in Figure 3f. Western blot analysis of ROX-induced Epo production in the body. (a) Epo protein expression in ROX-treated male rat. 1, Salivary gland; 2, thymus; 3, lung; 4, heart; 5, liver; 6, pancreas; 7, spleen; 8, adrenal gland; 9, kidney (cortex); 10, testes; and 11, epididymis. (b) Epo protein expressions in salivary gland, thymus, liver, spleen and kidney were compared in control and ROX-treated male rats. (c) Epo protein abundances in lung, pancreas, kidney, testis and epididymis were compared in control and ROX-treatedmale rats. (d) Epo protein expression in ROX-treated female rat. 1, Salivary gland; 2, thymus; 3, lung; 4, heart; 5, liver; 6, pancreas; 7, spleen; 8, adrenal gland; 9, kidney (cortex); and 10, ovary. (e) Epo protein expressions in salivary gland, thymus, liver, spleen, kidney and ovaries were compared in control and ROX-treated female rats. (f) Summarized data. Epo protein expression was normalized by measuring the β-actin expression. * indicates p < 0.05, n = 3-6. 1, Olfactory bulb; 2, cerebrum; 3, salivary gland; 4, thymus; 5, lung; 6, heart; 7, liver; 8, pancreas; 9, spleen; 10, adrenal gland; 11, kidney (cortex); 12, epididymis; and 13, ovary. a, Control and b, ROX. Molecules 2022, 27, x FOR PEER REVIEW 5 of 13 Figure 3. Effects of severe hypoxia on Epo protein expression in the body. (a) Epo protein expression in severe hypoxic male rat. (b) Epo protein expressions in salivary gland, thymus, liver, spleen and kidney were compared in control and severe hypoxic male rats. (c) Epo protein abundances in lung, pancreas, kidney, testis and epididymis were compared in control and severe hypoxic rats. (d) Epo protein expression in severe hypoxic female rat. (e) Epo protein expressions in salivary gland, liver, spleen, kidney and ovaries were compared in control and severe hypoxic female rats. (a,d) 1-10, the same as in Figure 2. (f) Summarized data. Statistical significance between control and hypoxic-treatment was obtained only in the kidneys. * indicates p < 0.05, n = 3-6. 1-13, the same as in Figure 2. a, control and b, severe hypoxia.
previously that, during severe hypoxia, Epo mRNA in the liver was only 1.5% of that seen in the kidneys.
Nonetheless, Epo mRNA expression in the liver increases during severe hypoxia (1, 36.7 ± 10.0*, 62.8 ± 12.0* and 183.0 ± 16.2* fold in 0, 1, 2 and 4 h after hypoxia, respectively, n = 3-5, * p < 0.05). In response to ROX, we observed an increase in the 35-38 kDa band density in lysates from the kidneys (lane R10) and liver (lanes 3 and 10, respectively). However, in response to ROX, while the 22 kDa band density increased in deglycosylated samples from the renal cortex (Figure 4e), no 22 kDa band was observed in deglycosylated samples from the liver (Figure 4e). The absence of a 22 kDa band in the deglycosylated liver lysates indicates that Epo was not produced by this tissue. These results also show that Epo production can be accurately quantified by measuring the abundance of the 22 kDa band by immunoblot of deglycosylated tissue samples. However, while ROX increased HIF1α mRNA in the kidneys, ROX reduced HIF1α mRNA in the liver (kidney: 1, 9.0 ± 5.6* and 14.8 ± 8.5* and liver: 1, 0.72 ± 0.06* and 0.76± 0.01* fold in control, R5 and R10, respectively, n = 3-5, Figure 4c). By the administration of the PHD inhibitor, ROX, PHD2 mRNA expression decreased in the renal cortex but increased in the liver (kidney: 1, 0.78 ± 0.22 and 0.25 ± 0.02* and liver:1, 5.38 ± 0.92* and 11.83 ± 1.31* fold in control, R5 and R10, respectively, n = 4-8, Figure 4d). We showed previously that, during severe hypoxia, Epo mRNA in the liver was only 1.5% of that seen in the kidneys.
In response to ROX, we observed an increase in the 35-38 kDa band density in lysates from the kidneys (lane R10) and liver (lanes 3 and 10, respectively). However, in response to ROX, while the 22 kDa band density increased in deglycosylated samples from the renal cortex (Figure 4e), no 22 kDa band was observed in deglycosylated samples from the liver (Figure 4e). The absence of a 22 kDa band in the deglycosylated liver lysates indicates that Epo was not produced by this tissue. These results also show that Epo production can be accurately quantified by measuring the abundance of the 22 kDa band by immunoblot of deglycosylated tissue samples.

Immunohistochemistry (IHC) of Epo Production by the Kidney
While Epo staining was seen in proximal and distal tubules in control rats, the label intensity did not change significantly with ROX administration (Figure 5g-i). Only a small increase in label intensity was observed in the proximal tubule following ROX administration (Figure 5g-i).

Immunohistochemistry (IHC) of Epo Production by the Kidney
While Epo staining was seen in proximal and distal tubules in control rats, the label intensity did not change significantly with ROX administration (Figure 5g-i). Only a small increase in label intensity was observed in the proximal tubule following ROX administration (Figure 5g-i).
Instead, ROX administration produced the greatest change in the Epo label within interstitial cells. While Epo staining was not detected in peritubular cells under basal conditions (Figure 5a,d), ROX increased the Epo label of the peritubular cells that surround the proximal tubule in a dose-dependent manner (Figure 5b,c,e,f). Under control conditions, Epo staining was found from the proximal tubule to the collecting duct (a). ROX increased Epo production in the peritubular cells around proximal tubules after 6 h (R10 > R5). Red arrowheads show Epo positive peritubular cells ((d-f) and red square of (a-c)). Epo staining was not increased in the distal tubules but slightly increased in the proximal tubules by ROX ((g-i) and yellow square of (a-c)). Scale bar: 20 µm (a-c) and 10 µm (d-i). PCT, proximal convoluted tubules; and CNT, connecting tubules.

Plasma Epo Concentration
Plasma Epo concentrations in control and ROX-treated rats are shown in Figure 6a. ROX (50 mg/kg) significantly increased the plasma Epo concentration from 1.2 ± 0.1 to 1072 ± 333 mIU/mL (n = 5-6, p < 0.001). The plasma Epo expression was also estimated using Western blotting. ROX increased the plasma Epo concentrations (1.4, 2690 and 3320 Under control conditions, Epo staining was found from the proximal tubule to the collecting duct (a). ROX increased Epo production in the peritubular cells around proximal tubules after 6 h (R10 > R5). Red arrowheads show Epo positive peritubular cells ((d-f) and red square of (a-c)). Epo staining was not increased in the distal tubules but slightly increased in the proximal tubules by ROX ((g-i) and yellow square of (a-c)). Scale bar: 20 µm (a-c) and 10 µm (d-i). PCT, proximal convoluted tubules; and CNT, connecting tubules.
Instead, ROX administration produced the greatest change in the Epo label within interstitial cells. While Epo staining was not detected in peritubular cells under basal conditions (Figure 5a,d), ROX increased the Epo label of the peritubular cells that surround the proximal tubule in a dose-dependent manner (Figure 5b,c,e,f).

Plasma Epo Concentration
Plasma Epo concentrations in control and ROX-treated rats are shown in Figure 6a. ROX (50 mg/kg) significantly increased the plasma Epo concentration from 1.2 ± 0.1 to 1072 ± 333 mIU/mL (n = 5-6, p < 0.001). The plasma Epo expression was also estimated using Western blotting. ROX increased the plasma Epo concentrations (1.4, 2690 and 3320 mIU/mL in control, R5 and R10, respectively). By Western blot of the plasma Epo, ROX dose-dependently increased both the glycosylated and deglycosylated Epo protein (Figure 6b). The abundance of the deglycosylated Epo protein in sample R10 was slightly less than that that of 498 pg of recombinant rat Epo protein.
Molecules 2022, 27, x FOR PEER REVIEW 8 of 13 mIU/mL in control, R5 and R10, respectively). By Western blot of the plasma Epo, ROX dose-dependently increased both the glycosylated and deglycosylated Epo protein (Figure 6b). The abundance of the deglycosylated Epo protein in sample R10 was slightly less than that that of 498 pg of recombinant rat Epo protein.

Discussion
The purpose of this study was to define the sites of Epo production both under basal conditions and in response to ROX or hypoxia. Therefore, we investigated Epo protein abundance under basal conditions and following either severe hypoxia or ROX treatment in many tissues, including the cerebrum, cerebellum, salivary glands, thymus, lungs, heart, liver, spleen, pancreas, adrenal glands, kidneys, testis, epididymis and ovaries.
While we detected Epo protein in the salivary glands, spleen, epididymis and ovaries under basal conditions, neither ROX nor severe hypoxia stimulated Epo production in those organs. Instead, our data showed that ROX and hypoxia stimulated Epo production only in the kidneys. Although severe hypoxia and ROX stimulated Epo mRNA expression in the liver, Epo protein production was not detected in the liver. Although ROX and hypoxia-induced Epo production was not observed other than in the kidneys, we cannot exclude the possibility that ROX produces side effects by targeting PHD in other tissues.
Epo and HIF2α mRNA expression increased not only in the kidneys but also in the liver. HIF1α mRNA expression was increased in the kidneys but decreased in the liver. PHD2 mRNA expression was decreased in the kidneys but increased in the liver. These data strongly suggest that the PHD inhibitor, ROX, stimulates Epo production through its effect in the kidneys but not in the liver.
To properly measure the Epo protein expression, we used both glycosylated and deglycosylated Epo expression. We showed the lower detection limit of deglycosylated 22 kDa bands compared with glycosylated 34-38 kDa bands using ROX-treated kidney lysates in this study (Figure 1c). Deglycosylated Epo production was not observed in the liver even with the increase of Epo mRNA expression. DNA methylation after birth may

Discussion
The purpose of this study was to define the sites of Epo production both under basal conditions and in response to ROX or hypoxia. Therefore, we investigated Epo protein abundance under basal conditions and following either severe hypoxia or ROX treatment in many tissues, including the cerebrum, cerebellum, salivary glands, thymus, lungs, heart, liver, spleen, pancreas, adrenal glands, kidneys, testis, epididymis and ovaries.
While we detected Epo protein in the salivary glands, spleen, epididymis and ovaries under basal conditions, neither ROX nor severe hypoxia stimulated Epo production in those organs. Instead, our data showed that ROX and hypoxia stimulated Epo production only in the kidneys. Although severe hypoxia and ROX stimulated Epo mRNA expression in the liver, Epo protein production was not detected in the liver. Although ROX and hypoxia-induced Epo production was not observed other than in the kidneys, we cannot exclude the possibility that ROX produces side effects by targeting PHD in other tissues.
Epo and HIF2α mRNA expression increased not only in the kidneys but also in the liver. HIF1α mRNA expression was increased in the kidneys but decreased in the liver. PHD2 mRNA expression was decreased in the kidneys but increased in the liver. These data strongly suggest that the PHD inhibitor, ROX, stimulates Epo production through its effect in the kidneys but not in the liver.
To properly measure the Epo protein expression, we used both glycosylated and deglycosylated Epo expression. We showed the lower detection limit of deglycosylated 22 kDa bands compared with glycosylated 34-38 kDa bands using ROX-treated kidney lysates in this study (Figure 1c). Deglycosylated Epo production was not observed in the liver even with the increase of Epo mRNA expression. DNA methylation after birth may cause the lack of Epo production after birth and under severe hypoxia or under PHD inhibition by ROX [32][33][34]. These data suggest the importance of measuring the Epo protein but not Epo mRNA expression.
In previous studies, we showed that Epo production is regulated not only by hypoxia but also by the renin-angiotensin-aldosterone system [35,37,38]. We previously showed that fludrocortisone and angiotensin II stimulated Epo production by the nephron, especially in the intercalated cells of the collecting duct [35,37,38].
In contrast, severe hypoxia and ROX stimulate Epo production through renal erythropoietin producing (REP) cells [34,[39][40][41][42][43][44] and increase the plasma Epo concentration. Our immunohistochemistry showed that both ROX and severe hypoxia increase Epo production by interstitial cells but not by tubular cells. The increase of plasma Epo concentration by ROX or severe hypoxia was higher than that by fludrocortisone or angiotensin II, showing that the Epo producing ability of REP cells is much higher than that of nephrons.
After 6 h, rats were injected with mixed anesthetic (0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam and 5.0 mg/kg of butorphanol), and blood was taken from the heart. The hypoxia-group rats were placed in 7% O 2 for 4 h, and control rats were placed in room air for 4 h before the injection of mixed anesthetic. Organs (the olfactory bulb, cerebral cortex, cerebellum, salivary glands, thymus, lungs, heart, liver, spleen, pancreas, adrenal glands, kidney cortex, epididymis and ovaries were taken after perfusing 20 mL of PBS from the heart. Our protocols were checked and approved by the Ethics Committee at Kitasato University Medical Center (25-2, 2018032, 2019029) and Kitasato University School of Medicine (2020-042).

Western Blot Analysis with Enzymatic Deglycosylation
Deglycosylation-coupled Western blot analysis was performed as described previously [26,31,32]. For the Western blot of plasma Epo, 10 µL of plasma was subjected to deglycosylation with and without PNGase as described below. Half of each sample was used for Western blot analysis. For the Western blot of tissue Epo, protein was extracted from organs using CelLytic MT (C-3228; Sigma-Aldrich, Burlington, VT, USA) plus protease inhibitor (05892970001, Roch, Basel, Switzerland) and used for Western blotting. In certain experiments, plasma from control and ROX-treated rats was also used.
Samples (lysates or plasma) were deglycosylated using N-glycosidase F (PNGase, 4450; Takara Bio, Kusatsu, Japan). We added 1 µL of 10% SDS to 10 µL of lysates samples and boiled for 3 min. Then, 11 µL of 2× stabilizing buffer was added, and the samples were vortexed. After the addition of 1 µL of PBS (glycosylated Epo) or PNGase (deglycosylated Epo), the samples were incubated in a water bath for 17-20 h at 37 • C. After the incubation, the samples were spun down, and the supernatant was collected and used for SDS-PAGE (10-20% gradient gel, 414893; Cosmo Bio, Tokyo, Japan).
Bands were visualized by the ECL Select Western Blotting Detection System (RPN2235; GE Healthcare Bio-Science AB, Uppsala, Sweden) and LAS 4000 (Fujifilm, Tokyo, Japan). Densitometric analysis was performed by Multi Gauge in LAS 4000. After measuring the Epo protein expression, the membrane was stripped (stripping solution, Wako, RR39LR, Tokyo, Japan) and reprobed with the antibody against β-actin (MBL, M177-3, Tokyo, Japan) for the normalization of the band. The molecular weight marker used was PagaRuler (26616, Thermo Scientific, Waltham, MA, USA).
The band at 35-38 kDa does not guarantee that the band is Epo. The band shift from 35-38 to 22 kDa by PNGase guarantees that the band is deglycosylated Epo protein. We showed that the 35-38 kDa and shifted to 22 kDa bands detected by sc-5290 represent Epo by LC/MS analysis using cut gels [35,36]. The assay sensitivity of glycosylated and deglycosylated Epo was compared with ROX-treated kidney lysates.
Images were obtained using an optical microscope (Axio Imager M2; Carl Zeiss, Oberkochen, Germany) with a digital camera (AxioCam 506, Carl Zeiss). Captured images were analyzed using an image analyzing system (ZEN 2, Carl Zeiss).

Plasma Epo Concentration Measurements
Plasma samples were taken from control and ROX-treated rats at 6 h after peritoneal injection. Plasma Epo concentrations were measured by CLEIA (SRL, Tokyo, Japan).

Statistical Analyses
Data are expressed as the mean ± SEM. Statistical significance was performed using Excel Statics (BellCurve, Tokyo, Japan). Statistical significance was analyzed using nonparametric analysis of the Kolmogorov-Smirnov test, Wilcoxon signed rank test or the Kruskal-Wallis test and multiple comparisons by the Shirley-Williams test. p < 0.05 was considered statistically significant.

Conclusions
In conclusion, our study showed that ROX and severe hypoxia increased Epo production only by the kidney interstitial cells by stimulating HIF1α and 2α expression and inhibiting PHD2 expression. Although ROX and severe hypoxia increased the Epo mRNA expression in the liver, Epo protein production was not observed.